A. Field of the Invention
The present invention relates generally to luciferases. In particular, the invention relates to proteins and peptides of luciferase enzymes that are functional to catalyse ATP dependent luminescence reactions producing light emission spectra with maximum intensities in the blue portion of the spectrum and assays utilizing such luciferases. In a further aspect, the invention relates to luciferases isolated from organisms of the genus Arachnocampa (Diptera).
B. Description of Related Art
The use of reporter molecules or labels to qualitatively or quantitatively monitor molecular events is well established. They are found in assays for medical diagnosis, for the detection of toxins and other substances in industrial environments, and for basic and applied research in biology, biomedicine, and biochemistry. Such assays include immunoassays, nucleic acid probe hybridization assays, and assays in which a reporter enzyme or other protein is produced by expression under control of a particular promoter. Reporter molecules, or labels in such assay systems, have included radioactive isotopes, fluorescent agents, enzymes and chemiluminescent agents.
Included in assay systems employing chemiluminescence to monitor or measure events of interest are assays that measure the activity of a bioluminescent enzyme, luciferase.
Light-emitting systems have been known and isolated from many luminescent organisms including bacteria, protozoa, coelenterates, molluscs, fish, millipedes, flies, fungi, worms, crustaceans, and beetles, particularly click beetles of genus Pyrophorus and the fireflies of the genera Photinus, Photuris, and Luciola. Additional organisms displaying bioluminescence are listed in WO/2000/024878 and 1999/049019. Also see Viviani, V. R., (2002) Cell. Mol. Life Sci. 59:1833-1850, which lists properties of bioluminescent terrestrial arthropods and insects.
In many of these organisms, enzymes catalyze monooxygenation, utilizing the resulting free energy to excite a molecule to a high energy state. Visible light is emitted when the excited molecule spontaneously returns to the ground state. This emitted light is called “bioluminescence.” Hereinafter it may also be referred to simply as “luminescence.”
Since the earliest studies, beetle luciferases (particularly that from the common North American firefly species Photinus pyralis) have served as paradigms for understanding of bioluminescence. The fundamental knowledge and applications of luciferases have typically been based on a single enzyme, called “firefly luciferase,”derived from Photinus pyralis. However, there are roughly 1800 species of luminous beetles worldwide. Thus, the luciferase of Photinus pyralis is a single example of a large and diverse group of beetle luciferases. It is known that all beetle luciferases catalyze a reaction of the same substrate, a polyheterocyclic organic acid (hereinafter referred to as “luciferin”, unless otherwise indicated), which is converted to a high energy molecule. It is likely that the catalyzed reaction entails the same mechanism in each case.
Beetle luciferases, including the so-called firefly luciferases, are members of the adenylate-forming superfamily of enzymes. Beetle luciferases catalyse a multistep reaction that has some similarities to the reactions catalysed by other adenylate-forming enzymes such as the acyl-CoA ligases, various other CoA ligases (e.g. 4-coumarate-CoA ligase) and peptide synthetases.
The first step of the reaction is the addition of an adenine monophosphate (adenylation) to the carboxylic carbon of D-luciferin to form luciferyl-AMP. ATP is the source of the AMP and the other product of the reaction is pyrophosphate. Thus, this reaction is “ATP dependent” or “dependent upon ATP”. Presumably hydrolysis of the pyrophosphate is used to drive the reaction forwards. Luciferyl-AMP is a mixed acid anhydride of acetic and phosphoric acids. It is relatively reactive because the adenylyl group is a good leaving group. In similar enzymes the first step is also adenylation of a carboxylate on the substrate molecule, whether it be acetate, a long-chain fatty acid, 4-coumarate, or an amino acid.
In the case of CoA utilising enzymes there is generally nucleophilic attack of the CoA sulphhydryl on the adenylated carboxylic acid resulting in conjugation of CoA to the substrate. In the case of beetle luciferases there is attack of molecular oxygen at the carbonyl, resulting in a highly energetic dioxetane intermediate, which subsequently decays releasing a photon, typically within the green-yellow portion of the spectrum (550-570 nm) plus carbon dioxide.
Luciferases possess features which render them particularly useful as reporter molecules for biosensing (using a reporter system to reveal properties of a biological system). Signal transduction in biosensors (sensors which comprise a biological component) generally involves a two step process: signal generation through a biological component, and signal transduction and amplification through an electrical component. Signal generation is typically achieved through binding or catalysis.
Conversion of these biochemical events into an electrical signal is typically based on electrochemical or caloric detection methods, which are limited by the free energy change of the biochemical reactions. For most reactions this is less than the energy of hydrolysis for two molecules of ATP, or about 70 kJ/mole. However, the luminescence elicited by luciferases carries a much higher energy content. Photons emitted from the reaction catalyzed by firefly luciferase (560 nm) have 214 Kj/einstein. Firefly luciferase converts chemical energy into light with high efficiency and extraordinary signal to noise characteristics. The quantum yield per molecule of D-luciferin is 0.88 (Seliger and McElroy 1960; Seliger and W. D 1960). This enzyme is therefore an extremely efficient transducer of chemical energy.
However, the known ATP dependent luciferases, e.g. the beetle luciferases, emit within a relatively narrow range of emission spectra. None of the known beetle luciferases emit with maximum emission intensities at wavelengths less than or equal to 530±5 nm (Viviani 2002; Nakatsu et al. 2006). Indeed, structural modifications to known luciferases permit lowering of the energy of emission spectrum (i.e. to maximum emission intensities at longer wavelengths), but not an increase in the energy of the emission spectrum (i.e. to shorter wavelengths). Further information on how structural modifications currently thought to modify emission spectrum may be found in Nakatsu et al. (2006).
Luciferases have been isolated directly from various sources and their cDNAs reported. See, for example: de Wet et al., Molec. Cell. Biol 7, 725-737 (1987); Masuda et al., Gene 77, 265-270 (1989); Nakatsu et al. (2006); and Wood et al., Science 244, 700-702 (1989)). With the cDNA encoding a luciferase in hand, it is entirely straightforward for the skilled to prepare large amounts of the luciferase by isolation from bacteria (e.g. E. coli), yeast, mammalian cells in culture, or the like, which have been transformed to express the cDNA. Alternatively, the cDNA, under control of an appropriate promoter and other signals for controlling expression, can be used in such a cell to provide luciferase (and ultimately bioluminescence catalyzed thereby) as a signal to indicate activity of the promoter. The activity of the promoter may, in turn, reflect another factor that is sought to be monitored, such as the concentration of a substance that induces or represses the activity of the promoter. Various cell-free systems that have recently become available to make proteins from nucleic acids encoding them, can also be used to make luciferases.
The ready availability of cDNAs encoding luciferases makes possible the use of the luciferases as reporters in assays employed to signal, monitor or measure genetic events associated with transcription and translation, by coupling expression of such a cDNA, and consequently production of the enzyme, to such genetic events.
For example, firefly luciferase has been widely used to detect promoter activity in eukaryotes and prokaryotes. Substrates required for the luminescence reaction, including a luciferin or other substrate, oxygen and ATP, are available or made readily available within living cells.
Multiple Reporter Assays
Mulitple, dual (or double) reporters are commonly used to improve experimental accuracy. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system. The term “multiple reporter” refers to the simultaneous expression and measurement of two or more individual reporter enzymes within a single system. When used together, two or more individual reporter enzymes may be termed “co-reporters”. Examples that currently benefit from multiple reporter assays include individual cells or cell populations (such as cells dispersed in culture, segregated tissues, or whole animals) genetically manipulated to simultaneously express two different reporter genes. Most frequently, the activity of one gene reports the impact of the specific experimental conditions, while the activity of a second reporter gene provides an internal control by which all sets of experimental values can be normalized. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by, for example, differences in cell viability or transfection efficiency. Other sources of variability, such as differences in pipetting volumes, cell lysis efficiency and assay efficiency, can be effectively eliminated. Thus, dual reporter assays often allow more reliable interpretation of the experimental data by reducing extraneous influences.
Cell-free reconstituted systems that may benefit from dual-enzyme reporter technology are cellular lysates derived for the simultaneous translation, or coupled transcription and translation, of independent genetic materials encoding experimental and control reporter enzymes. Immuno-assays may, likewise, be designed for dual-reporting of both experimental and control values from within a single sample.
Currently, genes encoding firefly luciferases (luc), Renilla luciferases, chloramphenicol acetyl transferase (CAT), beta-galactosidase (lacZ), beta-glucuronidase (GUS) and various phosphatases such as secreted alkaline phosphatase (SEAP) and uteroferrin (Uf; an acid phosphatase) have been combined and used as co-reporters of genetic activity. The following references provide representative examples of these various reporter genes used in combined form for the purpose of dual-reporting of genetic activity: luc and GUS: Leckie, F., et al., 1994; luc and CAT, and luc and lacZ: Jain, V. K. and Magrath, I. T., 1992; CAT and lacZ: Flanagan, W. M., et al., 1991. See also Promega Dual-Luciferase™ Reporter Assay system, the Dual-Glo™ Luciferase Assay System, described in its Technical Manual: Instructions for use of Products E2920, E2940, and E2980, revised 1/06, Part Number TM058; and Wood, K. V., (1998) The Chemistry of Bioluminescent Reporter Assays, Promega Notes 65, page 14, as well as Promega pGL3 Luciferase Reporter Vectors (available from Promega Corporation, Madison, Wis.) as well as U.S. Pat. Nos. 5,744,320 and 5,670,356.
The performance of any multiple reporter assay is limited by the characteristics and compatability of the constituent enzyme chemistries, and the ability to correlate the results respective to each. Disparate enzyme requirements or assay conditions may dictate that co-reporters may not be employed in an integrated, single-assay mixture or single-tube format. Ideally, a multiple reporter system would comprise at least two enzyme assays with compatible requirements, such as chemistries, temperatures, handling requirements, speed, sensitivity, detection instrumentation, etc.
In an attempt to meet the ideal requirements of a multiple reporter system, clones of different luciferases, particularly of a single genus or species, may be utilized together in bioluminescent reporter systems. The ability to distinguish each of the luciferases in a mixture, however, is limited by the width of their emissions spectra. Measurable variations in luminescence color from luciferases is needed for systems which utilize two or more different luciferases as reporters.
One example of luminescence color variation occurs in Pyrophorus plagiophthalamus, a large click beetle indigenous to the Caribbean. See, e.g. United States Patent Application 20030166905 published 4 Sep. 2003. The beetle has two sets of light organs, a pair on the dorsal surface of the prothorax, and a single organ in a ventral cleft of the abdomen. Four different luciferase clones have been isolated from the ventral organ and have been named LucPplGR, LucPplYG, LucPplYE and LucPplOR. The luciferin-luciferase reactions catalyzed by these enzymes produces light that ranges from green to orange.
Spectral data from the luciferase-luciferin reaction catalyzed by these four luciferases show four overlapping peaks of nearly even spacing, emitting green (peak intensity: 546 nanometers), yellow-green (peak intensity: 560 nanometers), yellow (peak intensity: 578 nanometers) and orange (peak intensity: 593 nanometers) light. As used herein, peak intensity: 546 nanometers, for example, means that the maximum emission of a luminescence spectrum produced by a luminescence reaction catalyzed by a luciferase occurs at or about 546 nanometers. The term “about” in this context refers to normal limits of precision in the measurement of the wavelength at which peak, or maximum intensity occurs (also known as lambda-max). Normal limits of precision are, for example, plus or minus 5 nanometers (i.e. ±5 nm).
Unfortunately, though the wavelengths of peak intensity of the light emitted by these luciferases range over nearly 50 nm, there is still considerable overlap among the spectra, even those with peaks at 546 and 593 nm. Increasing the difference in wavelength of peak intensity among the luminescence reactions employed would thus be highly desirable to obtain greater measurement precision in systems using two or more luciferases. In particular, a novel luciferase that catalyzes a luminescence reaction producing a maximum intensity equal to or less than about 530 nm would be highly desirable. Peak intensities of or less than about 530 nm are within the blue portion of the spectrum.
In one attempt to meet this need, the blue-emitting luminescent system of the sea pansy, Renilla reniformis, has been exploited in dual reporter systems with firefly luciferase. The luminescence of sea pansy (Renilla reniformis and closely related species) is in the blue spectrum, which offers advantages over green-yellow luminescence in some applications. The chemistry of the Renilla light reaction is unrelated to that of beetles and the reactive intermediate is generated by a different pathway from beetle luciferases. Specifically, Renilla luciferases catalyses the oxidation of coelenterazine to coelenteramide with emission of blue light at 480 nm. A dioxetane intermediate may be involved but adenylation does not occur. Renilla luciferase is evolutionarily unrelated to beetle luciferases or adenylate-forming enzymes. See, for example, U.S. Pat. Nos. 5,292,658 and 5,418,155.
Advantages and Limitations of Renilla Luciferase
The main advantage of Renilla luciferase/coelenterazine system as a reporter of gene expression is that it luminesces in the blue spectrum using different substrates from beetle luciferin. Renilla luciferase may therefore be used in double-labelling experiments with beetle luciferase/luciferin. See, e.g. U.S. Pat. No. 5,744,320. Nevertheless, in other aspects Renilla luciferase is inferior to beetle luciferases because the coelenterazine substrate exhibits a low level of non-enzymatic luminescence. This level of autoluminescence varies according to the hydrophobicity of the environment and together these two phenomena significantly limit the absolute sensitivity of assays.
Beetle luciferase does not suffer from this deficiency, presumably because the oxidiseable substrate (luciferyl-AMP) is never found free in solution but is synthesised, as described above, while tightly bound by the luciferase and is consequently very short-lived, with oxidation also occurring on the enzyme.
Additional shortcomings of Renilla luciferase is that it cannot be directly used in applications that involve ATP quantification, because the reaction does not utilise ATP. Also, Renilla luciferase is not amenable to continuous dual label assays in the presence of active or unquenched beetle luciferase.
Orfelia fultoni 
Orfelia fultoni, is a bioluminescent fly (Diptera) found in North America (Fulton, 1941). Its general biology is very similar to that of Australasian flies of the genus Arachnocampa, described in the next section. According to Viviani, Hasting et al. (Viviani, Hastings et al. 2002). Orfelia has a blue luminescence with lambda max=460 nm, a wavelength shorter than for any other insect-derived luminescence. Interestingly the Orfelia luminescence is not dependent on ATP, a feature that distinguishes it from the luminescence of all other insects, and is stimulated by some mild reducing agents. Viviani, Hastings et al. have partially characterised the luciferase of Orfelia biochemically (Viviani, Hastings et al. 2002).
Arachnocampa 
The presence of blue-emitting glowworms in sheltered habitats throughout Eastern Australia must have been well-known to indigenous Australians for tens of thousands of years. Closely related species occur in spectacular concentrations in New Zealand. The glowworms, which use their blue luminescence to attract prey into a sticky web, are the larvae of keroplatid flies. However, the first European descriptions misidentifed them as the larvae of beetles related to the European glow-worm Lampyris noctiluca (Coleoptera, Lampyridae). Currently recognized species (Baker 2004; Harrison 1966; Pugsley 1983) of Australasian glow-worms (Diptera: Keroplatidae: Arachnocampinae) are listed, in part, in Table 1.
TABLE 2NameGenus (subgenus) species (Authority)Arachnocampa (Campara) richardsae (Harrison)Arachnocampa (Arachnocampa) tasmaniensis (Ferguson)Arachnocampa (Campara) girraweenensis (Baker)Arachnocampa (Campara) gippslandensis (Baker)Arachnocampa (Campara) otwayensis (Baker)Arachnocampa (Campara) tropicus (Baker)Arachnocampa (Arachnocampa) buffaloensis (Baker)Arachnocampa (Campara) flava (Harrison)Arachnocampa (Arachnocampa) luminosa (Skuse)
According to (Shimomura, Johnson et al. 1966), the maximum of the Arachnocampa emission spectrum is 487±5 nm, which is in close agreement with the corrected emission spectrum presented by (Lee 1976) for A. richardsae. Viviani, Hastings et al. (2002) quote an emission maximum of 484 nm for A. flava. Lee (1976) also showed that the luminescence of Arachnocampa luciferase is stimulated by ATP and requires Mg++. The short wavelength emission suggests that Arachnocampa luciferase substrate is different from beetle luciferin (Wood 1983) and, indeed, beetle D-luciferin has not yet been demonstrated to stimulate the Arachnocampa luciferase (Lee 1976; Wood 1983; Viviani, Hastings et al. 2002).
Wood (1983) showed that luminescence in depleted cold extracts of Arachnocampa light organs could be regenerated by the addition of a heat-treated extract. Viviani, Hastings et al. (Viviani, Hastings et al. 2002) were able to extract some Arachnocampa luciferin using acidic ethyl acetate and performed a partial separation using TLC. No structural information is available regarding native Arachnocampa luciferin. According to Viviani, Hastings et al. (2002) the Arachnocampa luciferase has a molecular weight, estimated by gel filtration, of 36 kDa, i.e. approximately half the molecular weight of firefly luciferase, which is 62 kDa (Conti, Franks et al. 1996).